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| United States Patent |
6,251,951
|
|
Emerson
, et al.
|
June 26, 2001
|
Use of flavonoid and aromatic aldehydes as pesticides
Abstract
Methods and compositions based upon natural flavonoid and aromatic
aldehydes are provided, which find use as pesticides. The compositions are
effective against pathogenic fungi and insects at concentrations which are
not phytotoxic to the treated host plant. Infestations of a variety of
plant parts can be treated, including those of leaves and roots.
Susceptible organisms include rust, powdery mildew and aphids.
| Inventors:
|
Emerson; Ralph W. (Davis, CA);
Crandall, Jr.; Bradford G. (Davis, CA)
|
| Assignee:
|
Proguard, Inc (Suisun, CA)
|
| Appl. No.:
|
479623 |
| Filed:
|
June 7, 1995 |
| Current U.S. Class: |
514/701; 424/405; 424/406; 424/407; 514/693 |
| Intern'l Class: |
A01N 035/02 |
| Field of Search: |
424/408,417,405-407,409,419-421,195.18,742,770,775
514/701,693
|
References Cited
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| 5639794 | Jun., 1997 | Emerson | 514/699.
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| WO93/05159 | Mar., 1993 | WO.
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| WO94/27434 | Dec., 1994 | WO.
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| WO95/15082 | Jun., 1995 | WO.
| |
| WO 96/20596 | Jul., 1996 | WO.
| |
| WO 96/41528 | Dec., 1996 | WO.
| |
Other References
Mahmoud--Antifungal-Essential Oils: Letters. App.Micro. 19; 110-113 1994.*
Horst et al Plant Disease vol 76 #3 1992.*
Greef et al Mitt.Biol.Bundesanst.Landrorstwirtsch #266, p. 220 1990.*
Bullerman et al.J.Food Science 42 (4) 1977 pp.1107-1109.*
Keene et al Physiol. Plant Path. vol14(3) pp. 265-280 1979.*
Hagiwara et al: Hokkaido Sochi Kenkyukaiho pp. 74-77, 27, 1993.*
Yuan et al Fundamental & Applied Toxicology 20:83-87, 1993.*
Mawo et al Outlook on Agriculture vol 7 #5 p. 231-235 1973.*
Vaughn et al: J Agric. Food Chem(1994), 42 (1),200-3 Antifungal Activity of
Natural Compounds--.*
Gorris et al Brighton Crop Prot. Conf-Pests Dis. 1994;vol. 1; 307-12 :
Control of Fungal Storage Cleaner of Potato--.*
Sitaramaiah et al. Chemical Abstracts (1982) 96(13) (Abstract No. 99381).
Lamb C. J. et al. Bio/Technology (1992) 10:1436-1445.
Bowles & Miller, J. Food Protection (1993) 56: 788-794.
Casey & Dobb, Enzyme Microb. Technol. (1992) 14: 739-747.
Yuan et al., Fundamental & Applied Toxicol. (1993) 20: 83-87.
Ishibashi & Kubo, Proc. Assc. Plants (1987) 33: 122-125.
King, Agriculture Handbook (1954) 69: 1-397 (relevant pages attached).
Matsumoto Microbiology Laboratory, Antimicrobial Test of Avion-M (1982)
57-07 (full cite not available).
Frear, Chemistry of Insecticides and Fungicides (1942 13 184-191.
Ishibashi et al, Nematicidal effect of cinnamic aldehyde on root-knot
nematode, Meloidgyne incognita (1987) 33: 122-125.
Hagiwara et al, Effect of cinnamaldehyde on the growth of Rhizoctonia
solani (AG2-2 IIIB) and development of brown patch disease on bentgrass
(1993) 27: 74-77.
Ohtsuka et al, Effects of Abion CA chemicals on vegetable diseases (1983)
29: 48-51.
|
Primary Examiner: Levy; Neil S.
Attorney, Agent or Firm: Venter; Barbara Rae
Rae-VenterLaw Group, PC
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation-in-part of U.S. Ser. Nos. 08/366,973,
Ser. No. 08/367,082 filed on Dec. 30, 1994, which is related to two
copending applications filed on Dec. 30, 1994, namely and U.S. Ser. No.
08/366,974, which disclosures are herein incorporated by reference.
Claims
What is claimed is:
1. A method for protecting a chrysanthemum from infestation and attack by
powdery mildew, said method comprising:
contacting one or more parts of said chrysanthemum with a formulation
comprising an amount of from 2.5 to 50 g/l of cinnamic aldehyde, wherein
aid amount is sufficient to provide at least 70% inhibition of the growth
of aid powdery mildew, wherein said formulation has a phytoxicity rating
of 1 or less for said chrysanthemum, and wherein said formulation does not
include an anti-oxidant whereby said chrysanthemum is protected from
infestation and attack by said powdery mildew.
2. A method for protecting a bell pepper, a lettuce or a tomato plant from
infestation and attack by powdery mildew, said method comprising:
contacting one or more parts of said plant with a formulation comprising
amount of from 2.5 to 50 g/l of cinnamic aldehyde, wherein said amount is
sufficient to provide at least 70% inhibition of the growth of said
powdery mildew, wherein said formulation has a phytotoxicity rating of 1
or less for said plant, and wherein said formulation does not include an
anti-oxidant, whereby said plant is protected from infestation and attack
by said powdery mildew.
3. A method for protecting a cantaloupe or a grape plant from infestation
and attack by powdery mildew said method comprising:
contacting one or more parts of said plant with a formulation comprising
amount of from 2.5 to 50 g/l of cinnamic aldehyde, wherein said amount is
sufficient to provide at least 70% inhibition of the growth of said
powdery mildew, wherein said formulation hag a phytotoxicity rating of 1
or less for said plant, and wherein aid formulation does not include an
anti-oxidant whereby said plant is protected from infestation and attack
by said powdery mildew.
4. A method for protecting a citrus tree from infestation and attack by
powdery mildew, said method comprising:
contacting one or more parts of said citrus tree with a formulation
comprising an amount of from 2.5 to 50 g/l of cinnamic aldehydes, wherein
said anoint is sufficiently to providing at least 70% inhibition of the
growth of said powdery mildew, wherein said formulation has a phytoxicity
rating of 1 or legs for said rose, and wherein said formulation does not
include an anti-oxidant, whereby said citrus tree is protected from
infestation and attack by said powdery mildew.
5. A method for protecting a rose from infestation and attack by powdery
mildew, said method:
contacting one or more parts of said rose with a formulation comprising an
amount of from 2.5 to 50 g/l of cinnamic aldehyde, wherein said anoint is
sufficient to provide at least 70% inhibition of the growth of ,aid
powdery mildew, wherein said formulation has a phytotoxicity rating of 1
or less for said rose, and wherein said formulation does not include an
anti-oxidant, whereby said rose is protected from infestation and attack
by said powdery mildew.
6. A method for protecting a cabbage plant from infestation and attack by
powdery mildew, said method comprising:
contacting one or more parts of said cabbage plant with a formulation
comprising an amount of from 2.5 to 90 g/l of cinnamic aldehyde, wherein
said amount is sufficient to provide at least 70% inhibition of the growth
of said powdery mildew, wherein said formulation has a phytotoxicity
rating of 1 or less for said cabbage plant, and wherein said formulation
does not include an anti-oxidant, whereby said cabbage plant is protected
from infestation and attack by said powdery mildew.
7. A method for protecting a bent grass or a turf grass plant from
infestation and attack by powdery mildew, said method comprising:
contacting one or more parts of ,aid plant with a formulation comprising an
amount of from 2.5 to 50 g/l of cinnamic aldehyde, wherein said amount is
sufficient to provide at least 70% inhibition of the growth of said
powdery mildew, wherein aid formulation has a phytotoxicity rating of 1 or
less for said plant and wherein said formulation does not include an
anti-oxidant whereby said plant is protected from infestation and attack
by aid powdery mildew.
8. A method for protecting a grass from infestation and attack by
Sclerotinia homoeocarca, said method comprising:
contacting one or more parts of said grass with a formulation comprising an
amount of from 2.5 to 50 g/l of cinnamic aldehyde, wherein said amount is
sufficient to provide at least 70% inhibition of the growth of Sclerotinia
homoeocarca, wherein said formulation has a phytotoxicity rating of 1 or
less for said grass, and wherein said formulation does not include an
anti-oxidant, whereby said grass is protected from infestation and attack
by said Sclerotinia homoeocarca.
9. A method for protecting a rose from infestation and attack by at least
one pathological organism, said method comprising:
contacting one or more parts of said rose with a formulation comprising an
amount of from 2.5 to 50 g/l of coniferyl aldehyde, wherein said amount is
sufficient to provide at least 70% inhibition of the growth of at least
one pathological organism selected from the group consisting of powdery
mildew and rust, wherein said formulation has a phytotoxicity rating of 1
or less for said rose, and wherein said formulation does not include an
anti-oxidant, whereby said rose is protected from infestation and attack
by said at least one pathological organism.
10. A method for protecting a rose from infestation and attack by powdery
mildew, said method comprising:
contacting one or more parts of said rose with a formulation comprising an
amount of from 2.5 to 50 g/l of cinnamic aldehyde and from 2.5 to 50 g/l
of coniferyl aldehyde, wherein said amount is sufficient to provide at
least 70% inhibition of the growth of said powdery mildew, wherein said
formulation has a phytotoxicity rating of I or less for said rose, and
wherein said formulation does not include an additional anti-oxidant,
whereby said rose is protected from infestation and attack by said powdery
mildew.
11. A method for protecting a rose from infestation and attack by one or
more pathological organism, said method comprising:
contacting one or more parts of said rose with a formulation comprising an
amount of from 2.5 to 50 g/l of cinnamic aldehyde, wherein said amount is
sufficient to provide at least 70% inhibition of the growth of a
pathological organism selected from the group consisting of powdery mildew
and rust, wherein said formulation has a phytotoxicity rating of I or less
for said rose, and wherein said formulation does not include an
anti-oxidant, whereby said rose is protected from infestation and attack
by said one or more pathological organism.
12. A method for protecting a grape from infestation and attack by powdery
mildew, said method comprising:
contacting one or more parts of said grape with a formulation comprising an
amount of from 2.5 to 50 g/l of cinnamic aldehyde, wherein said amount is
sufficient to provide at least 70% inhibition of the growth of aid powdery
mildew, wherein said formulation has a phytotoxicity rating of 1 or less
for said grape, and wherein said formulation does not include an
anti-oxidant, whereby said grape is protected from infestation and attack
by said powdery mildew.
13. The method according to any one of claims 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11 or 12, wherein said formulation further comprises saponin.
14. The method according to any one of claims 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11 or 12, wherein said contacting is by spraying.
15. The method according to any one of claims 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11 or 12, wherein said formulation is an aqueous formulation.
16. The method according to any one of claims 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11 or 12, wherein said one or more parts of said plant are leaves.
Description
INTRODUCTION
1. Technical Field
The present invention is related to the biocontrol of plant pathogens
through nutritional mediation. The invention is exemplified by the use of
cinnamic aldehyde and coniferyl aldehyde to control growth of fungi and
parasitic insects, including sap sucking insects which colonize the
surfaces of plant parts and tissues.
2. Background
The surfaces of plant parts such as roots and leaves are colonized by a
variety of organisms, many of which are dependent upon the host plant as a
source of nutrients. The colonizing organisms include pathogenic fungi and
sap-sucking insects; both groups are capable of inflicting severe damage
to the host plant, including stunting the growth of the host plant and
decreasing plant productivity, to killing the host plant.
Fungi pathogenic for plants are many and diverse. They occur in most groups
of fungi. A few, such as rusts, Uredinales, and powdery mildew and downy
mildew, Erysiphacea and Peronosporacea, are obligate parasites. Generally,
a particular rust or mildew is associated with specific host plants which
elaborate nutrients required by the pathogen. As an example, rust, caused
by Phragmidrium mucronatrum, is an important fungal disease associated
with roses; it produces bright orange pustules on the underside of rose
leaves and pale yellow spots on the top. Powdery mildew, caused by
Sphaerotheca pannosa (Wallr. ex. Fr.) Lev var. rosae Woronichine also is
associated with roses and is probably the most widely distributed and
serious disease of glasshouse, garden, and field-grown roses alike.
Pathogenic insects which infest plants include those insect species which
are symbiotic with bacteria, such as aphids, leaf hoppers, and white fly;
the host insect cannot survive without the symbionts. As an example,
aphids (homoptera) possess symbiotic bacteria of the genus Buchnera in
cells called mycetocytes within the hemocoel. The bacteria are transmitted
directly from the maternal aphid to her offspring and aposymbiotic aphids
do not occur naturally. The bacteria may provide lipids which are required
for embryogenesis of the host insect but which are absent or in low
concentrations in phloem sap in plants infected by the insects.
The plant pathogens include the grape phylloxera (Daktulosphaira
vitifoliae), an aphid-like insect, and nematodes. Phylloxera is native to
the United States east of the Rocky Mountains, where it lives on native
wild species of grapes, which have evolved resistance to the feeding of
the insect. The European grape (Vitis vinifera), which is used to produce
wine, evolved in western Asia and has no resistance to phylloxera. Stem
and bulb nematode (Ditylenchus dipsaci) has been recorded from all the
major agricultural regions in California This wide distribution probably
reflects its spread on such infested planting material as garlic cloves.
Wherever such infested material is grown, the nematode may be introduced.
Ditylenchus dipsaci can be found parasitizing a wide range of cultivated
and wild plants. Nematodea produce galls in infected tissue. In addition
to the disturbance caused to plants by the nematode galls themselves,
damage to infected plants is increased by certain parasitic fungi, which
can easily attach the weakened root tissues and the hypertrophid,
undifferentiated cells of the galls. Moreover, some fungi, for example,
Pythrium, Fusarium, and Rhizoctonia, grow and reproduce much faster in the
galls than in other areas of the root, thus inducing an earlier breakdown
of the root tissues.
A variety of pesticide compositions are used for controlling plant
pathogens. For example, protective fungicidal sprays on a 6-7 day schedule
for both rust and powdery mildew when environmental conditions favor
disease development are the typical means of control. Two frequently used
systemic fungicides are benomyl and triforine. However, the cost of
fungicides for control of powdery mildew is high: in cut rose crops the
cost of treatment in the State of California is several million dollars a
year.
The older fungicides include inorganic compounds such as copper and sulphur
and the organic protectants such as thiram, captam, maneb, and
chlorotholonil. These compounds act only at the surface of the plant and
must be present at or before appearance of the fungal pathogen in order to
prevent infection. These older fungicides are multisite inhibitors; i.e.,
they affect many metabolic activities in a fungus. The newer fungicides
tend to be highly effective organic systemics such as benzimididazoles,
sterol biosynthesis inhibitors, carboxanilides, and phenylamides which act
internally as well as at the plant surface. In contrast to the older
surface protectants, the systemic fungicides are generally effective at
much lower dosages and can cure established fungal infections, a critical
factor in disease management. The systemic fungicides usually act at a
single target site in the fungus, interfering with specific metabolic
processes that are necessary for production of all new cell material
required for growth, maintenance, and virulence of the fungal organism.
These preparations typically are effective only against fungal pathogens.
Current methods of chemical control for certain above-ground pests (e.g.,
spider mite, aphids, silverleaf white fly, leaf hoppers) include those
which combine two insecticides from different chemical classes, for
example, combining a synthetic pyrethroid with an organophosphate or
organochlorine insecticide. Soil fumigants have been a popular treatment
for soil pests (nematodes, phylloxera). Use of certain highly effective
types of insecticides and fumigants has sharply decreased in recent years
due to cancellations of public regulatory agency registrations, or
refusals of re-registrations, of products.
The wide-spread use of pesticides has resulted in the development and
evolution of resistant pathogens, as well as growing environmental and
health care concerns. A highly visible ecological-environmental activist
community and public regulatory agencies have resulted in fewer and fewer
pesticide registrations and, consequently, less related research and
development.
The use of flavonoid and aromatic aldehydes for treatment of both fungal
and other pathogens has been reported. However the preparations used have
been reported to require the use of expensive antioxidants, and at the
concentrations used, would be expected to be phytotoxic to the host plant.
Such formulations also are reported to require multiple applications to
ensure continued protection of the host plant. It therefore is of interest
to identify and/or develop, "biorational" fungicides which have lower
animal and environmental toxicities and which also do not exhibit
significant phytotoxicity at the concentrations used to control pathogenic
fungi and insects.
3. Relevant literature
A method of protecting crops from attack of insect pests, microorganisms
and pathogenic microbes using a composition comprising cinnamic aldehyde
and requiring an antioxidant is disclosed in U.S. Pat. No. 4,978,686.
Protection of crops against pathogenic microorganisms and insect pests by
applying an aqueous composition containing a cinnamaldehyde is disclosed
in French patent application 2529755. U.S. Pat. No. 2,465,854 describes an
insecticidal composition containing a cinnamic aldehyde derivative.
Control of Verticillium using cinnamaldehyde in the substrate in which
mushrooms are grown is disclosed in U.S. Pat. No. 5,149,715.
Reweri, et al. describe induction of systemic resistance to powdery mildew
in cucumber by phosphates. Biol. Agic. and Hort. (1993) 9:305-315. Horst
and Kawamato disclose the effect of sodium bicarbonate and oils on the
control of powdery mildew and black spot on roses. Plant Disease, March
1992, p.247. Sodium bicarbonate and severely solvent refined light
paraffinic petroleum oil have been used to control black spot and powdery
mildew. Ziv et al., Hort. Science (1993) 28:124-126.
Elad et al. disclose the effect of film-forming polymers on powdery mildew
of cucumber. Phytoparasitica (1989), 17:279-288. Hagiladi and Ziv disclose
the use of antitranspirant for the control of powdery mildew in the field.
J. Environ. Hortic. (1986), 4:69-71. Macro, et al. disclose control of
powdery mildew of roses with antitranspirant coating polymers.
Phytoparasitica (1994) 22:19-29. Paulus and Nelson disclose use of
flusilarzol, myciobutanil, fenarimol, pentonazote, and diniconazole for
controlling powdery mildew and rust in roses. Calif. Agric. 1988, 42:15.
U.S. Pat. No. 4,402,950 describes the deactivation of viruses inside lining
human and animal organisms by application of a terpene obtainable from
aromatic plants by steam application. The terpenes cited are: black pepper
oil, cinnamon flour oil, cardamon oil, linallyl acetate, cinnamic
aldehyde, safrol, carvon and cis/trans citrao. U.S. Pat. No. 4,477,361
describes a cinnamic compound containing an anti-microbial surfactant
which is rendered substantive to the surface being washed.
References relating to anti-microbial properties of various saponins either
alone or in combination with other agents include the following:
JP2157205, DE3724595, JP61065802, JP61007290.
SUMMARY OF THE INVENTION
The present invention provides a method for controlling pathogenic
organisms on plants, as well as seeds, seedlings and plants substantially
free of plant pathogens through nutritional mediation. The method includes
the step of contacting one or more parts or tissues of a diseased plant or
a plant susceptible to attack by pathogens with an antipathogenic agent in
an amount sufficient to control growth of target pathogenic organisms. The
growth modulating product has a formula shown in (1) below:
##STR1##
wherein R represents --CH.sub.2 OH or --CHO; n is an interger from 0 to 3;
and each R.sup.1 independently represents OH or an organic substitutent
containing from 1 to 10 carbon atoms and from 0 to 5 heteroatoms, wherein
the total number of carbon and heteroatoms in all R.sup.1 substitutents of
said compound is no more than 15. These compounds include natural
compounds such as cinnamaldehyde, coniferyl aldehyde, and closely related
compounds. The method finds use in treating ornamentals and agricultural
crops for pathogenic organisms.
DESCRIPTION OF SPECIFIC EMBODIMENT
Seeds, seedlings, plants, and plant parts such as fruit substantially free
of pathogenic organisms such as fungi and sapsucking insects are provided
together with a method to biocontrol pathogen infestations on plants using
flavonoid and aromatic aldehydes. By "biocontrol" is intended control of
plant pathogens via direct antipathogenic activity and/or induced
resistance of the host plant to pathogen infestation. A fungus and/or
insect colonizing surface of a plant part such as a leaf, root, or flower
part, or a tissue such as xylem or phloem, is contacted with a natural
product. By "colonizing" is intended association of a microorganism or
insect with a plant part or tissue from which the pathogen derives
nutrients, typically essential nutrients such as amino acids, particularly
methionine. By "natural product" is intended an organic compound of
natural origin that is unique to one organism, or common to a small number
of closely related organisms, and includes secondary metabolites of fungi
and chemicals produced by plants. The natural products can be isolated
from a natural source, be wholly or partly synthetic, or be produced by
recombinant techniques.
The method of the subject invention is carried out by adding an effective
pathogen-inhibiting amount of a compound of the invention to a plant host
or to the substrate in which it is growing or is to be growing. The amount
of antipathogenic agent that is applied either to the plant itself or to
the rhizosphere will depend upon the degree of infestation and to some
extent upon the formulation and the specific compounding used and
therefore must be empirically determined for best results. By
"antipathogenic" is intended a pesticide, i.e. a formulation which is
effective for controlling the growth of pathogens and can involve killing
the pathogen and/or slowing or arresting its proliferation. Pathogens
include insects, fungi and other microorganisms which negatively affect
the plants which they colonize.
The compositions and methods of the subject invention offer several
advantages over existing compositions and methods. Although a flavonoid
aldehyde, cinnamic aldehyde, has been reported to exhibit antifungal
properties, it has not previously been used on plants in the absence of an
anti-oxidant. As an example, U.S. patent application Ser. No. 4,978,686,
discloses that an antioxidant is required for use with cinnamic aldehyde
for a composition which is used for application to crops. Anti-oxidants
are expensive, accordingly significant cost benefits are realized with the
subject formulation. In addition, a single application of cinnamic
aldehyde is sufficient for long term protection of the plant host from
pathogenic organisms, including both rust and powdery mildew, and is
effective at lower concentrations than has been reported previously.
Phytotoxicity of the formulation also is decreased due to the lower
concentrations of aldehyde which are used. The long term control of
pathogenic organisms results in a healthier plant and an improved yield of
produce by the host plant as compared to untreated plants; the lower
concentrations and single dose of antipathogenic agents decrease the
likelihood of damage to the plant or its crop as well as decrease the
likelihood of any adverse side effects to workers applying the pesticide,
or to animals, fish or fowl which ingest the tissues or parts of treated
plants.
More and more pesticides are not being re-registered by public regulatory
agencies. For example, the Delaney clause has recently put into question
the future of many pesticides. With limited pesticide availability,
certain applications, such as the treatment of phylloxera on grape roots
where translocation is important in the treatment and control of the
insect pest and related fungal pathogens, will be problematic.
The subject formulations also provide for effective control of both fungi
and insects, eliminating the need for application of multiple agents. In
particular situations, such as where an insect damages a plant part or
tissue and a secondary fungal disease develops, this aspect of the
invention is particularly advantageous. The subject formulation is as
shown in formula (1) above. A preferred formulation is shown in formula
(2) below:
##STR2##
wherein R.sub.1 represents --CHO, R.sub.3 represents --H--OH or an organic
substituent containing from 1 to 10 carbon atoms and from 0 to 5
heteroatoms; and R.sub.2 represents --H a methoxy group or organic
substituent containing from 1 to 10 carbon atoms, and from 0 to 5
heteroatoms. Of particular interest are aromatic aldehydes. Examples of
aromatic aldehydes of use in the present invention are cinnamic aldehyde
((3) below):
##STR3##
and coniferyl aldehyde ((4) below):
##STR4##
A number of the aromatic and aliphatic aldehydes which find use in the
subject invention, such as benaldehyde, acetaldehyde, cinnamaldehyde,
piperonal, and vanillin are generally regarded as safe (GRAS) synthetic
flavoring agents (21 CFR .sctn.172.515). These compounds have been
reported to have inhibitory activity against C. botulinum spore
germination. Bowles and Miller, G. Food Protection (1993) 56:788-794. The
general formula of these compounds is shown above as (1).
The aromatic and aliphatic aldehydes of the subject invention may be
prepared by various synthetic methods known to those skilled in the art.
For example, see, J. March, ed., Appendix B, Advanced Organic Chemistry:
Reactions, Mechanisms, and Structure, 2nd Ed., McGraw-Hill, New York,
1977. Cinnamaldehyde may be prepared synthetically, for example, by
oxidation of cinnamyl alcohol (Traynelis et al., J. Am. Chem. Soc. (1964)
86:298) or by condensation of styrene with formylmethylaniline (Brit.
patent 504,125). The subject aldehydes may also be obtained by isolation
from natal sources. For example, cinnamaldehyde may be isolated from
woodrotting fungus, Stereum subpileatum. Birkinshaw et al., Biochem. J.
(1957) 66:188.
The compounds may be used either alone or in combination with other active
or inactive substances and may be applied by spraying, pouring, dipping,
in the form of concentrated liquids, solutions, suspensions, powders and
the like, containing such concentration of the active compound as is most
suited for a particular purpose at hand. They may be applied, for example,
in the form of dilute solution, in a suitable solvent directly to the
rhizosphere either as part of an irrigation schedule or as a separate
application. For use as a foliar spray, although the aldehyde can be
formulated alone, it can be rendered substantive by including an
emulsifier such as Tween 80. Other detergents which can be used include
anionic detergents such as those described in U.S. patent application Ser.
No. 4,978,686. Other compounds which can be used alone or in conjunction
with detergents to increase the substantive properties of the formulation
include saponins from any of a variety of sources. Generally, detergents
and other agents used in the formulation do not detract from the pesticide
properties of the aromatic aldehydes but do increase the substantive
properties of the formulation (see for example, U.S. patent application
Ser. No. 4,477,361) and may improve the pesticide properties (see below).
Additional components such as an aqueous preparation of a salt of a
polyprotic acid such as sodium bicarbonate, sodium sulfate, sodium
phosphate or sodium biphosphate can be included in the formulation, to
increase the antifungal properties of the formulation. The resulting
emulsion is diluted to an appropriate concentration for use.
In a preferred embodiment, the formulation includes cinnamic aldehyde
and/or coniferyl aldehyde in a formulation containing Tween 80 as an
emulsifier and sodium bicarbonate. The preferred formulation for treating
powdery mildew, rust and spores, as well as aphids is an emulsion which
contains cinnamic aldehyde and/or coniferyl aldehyde, 0.001% to 10% by
weight, the salt of a polyprotic acid, 4% to 12% by weight, an emulsifier
2% to 4%, and the balance water. Generally, the total amount of
aldehyde(s) present in the formulation is 5% or less. The formulations are
effective and stable without the use of antioxidants, although particular
aldehydes may have inherent antioxidant properties, for example, coniferyl
aldehyde. Stability of the formulation can be evaluated by a variety of
methods, including accelerated tests in which a formulation of interest is
exposed to elevated temperatures over a set time. Samples of the
formulations are taken at regular intervals and analyzed chemically by
methods known to those skilled in the art to determine the rate and nature
of degradation.
The most effective amount for compositions including compounds of formula
(3) and/or formula (4) as well as the amount of other compounds of formula
(1) which may find use can be determined using protocols such as those
described in the Examples. Generally, an effective growth modulating
amount of one or more compounds of formula (2) is 0.01 g/l to 25 g/l.
These protocols also can be used to optimize each formulation for specific
pathogens using any of the compounds encompassed by formula (1) as well as
for use on specific plants to minimize phytotoxicity while maximizing the
antipathogenic effect of the formulation.
In some instances, the efficacy of the formulation can be increased by
adding one or more other components, i.e., a compound other than a
compound of formula (1), to the formulation where it is desirable to alter
particular aspects of the formulation. As an example, it may be desirable
for certain applications to decrease the phytotoxicity effect
(phytotoxicity rating of 2 or less, with 1 or less preferred, below) or to
increase the antipathogenic effect of the formulation (mean disease
resistance of 60% or better, with a least about 70% or greater preferred,
see below) or both. It is preferable that the additional component(s)
minimize phytotoxicity while increasing the antipathogenic effect of the
formulation. Of particular interest is the use of a component(s) which is
a synergist to increase the mean disease resistance while minimizing the
phytotoxic effect as related to a particular formulation. By "synergist"
is intended a component which, by virtue of its presence, increases the
desired effect by more than an additive amount. The concentration of one
or more of the other formulation ingredients can be modified while
preserving or enhancing the desired phytotoxic and antipathogenic effect
of the formulation. Of particular interest is the addition of components
to a formulation to allow for a reduction in the concentration of one or
more other ingredients in a given formulation while substantially
maintaining efficacy of the formulation. Combination of such a component
with other ingredients of the formulation can be accomplished in one or
more steps at any suitable stage of mixing and/or application.
Preferred additional components include saponins. Saponins are a class of
compounds, each consisting of a sapogenin portion and a sugar moiety. The
sapogenin may be a steroid or a triterpene and the sugar moiety may be
glucose, galactose, a pentose, or a methylpentose. S. Budavari, ed., The
Merck Index, 11th ed., Merck & Co., Inc., Rahway, N.J., 1990, p. 1328. The
saponins for use in the present invention can be produced and/or isolated
from various plant parts including fruit, leaf, seed and/or root, using
means known in the art, from a variety of sources including the various
plants known to produce them, ranging from yucca, quillaja, agave,
tobacco, licorice, soybean, ginseng and asparagus to aloe woods. Saponins
for use with the present invention are preferably non-toxic to humans and
higher animals. Most preferably the saponin for use in the present
invention is non-toxic food grade, the source being from yucca plants.
Even more preferred are the saponins from Yucca schidigera or Y. valida
and their equivalents. The saponins are generally prepared by a cold press
extrusion process and the resulting liquid extract analyzed by HPLC for
saponin concentration. The yucca fiber also can be used; it is typically
sundried, mulled and sized by screening.
A variety of rally related saponins are known, the most variable structural
feature being the glycosylation pattern. Saponins also may contain
additional modifications, such as the sarasaponins which are saponins with
a steroid attached, and saponin structure can be modified by any number of
enzymatic, chemical and/or mechanical means known in the art. Nobel, Park
S., Agaves, Oxford Univ. Press, New York, 1994, pp. 42-44. Accordingly,
derivatives of these compounds which produce a formulation having the
desired antipathogenic and/or phytotoxic effect are considered equivalents
of the invention. Depending on its structure, a given saponin can have a
particular pesticidal property and lend use with the present formulations.
Generally an effective amount of saponin is of the range 0.01 to 0.1% and
most preferably about 0.01% v/v aqueous solution of 10.degree. brix
saponin extract.
For applications where the formulation is to be used to prepare the ground
or other growth substrate for planting of host plants susceptible to
particular pathogens, or to apply to an already infested growth substrate,
the formulations of the subject invention can be added directly to the
rhizosphere or the substrate or they can be bound to a solid support or
encapsulated in a time release material. Where a solid carrier is used,
materials which can lead to oxidation of the active aldehydes should be
avoided. Examples of delivery systems which can be used include
starch-dextran, and the like. See Yuan et al., Fundamentals and Applied
Toxicology (1993) 20:83-87 for other examples of appropriate materials.
In addition to the specific compounds of the formulas (1), (2), (3) and (4)
set forth above, derivatives of any of these compounds that produce a
compound of the formula identified above upon action of a biological
system on the derivative are considered to be equivalent to compounds of
the invention. Thus application of precursor compounds to plant parts or
tissues would be equivalent to the practice of the present invention.
Biological conversion of precursor compounds into aromatic aldehydes is
described in, for example, U.S. patent application Ser. No. 5,149,715 and
references cited therein. So also Casey and Dobb Enzyme Microb. Technol.
(1992) 14:739-747.
The method of the present invention is carried out by introducing into a
target pathogenic organism a sufficient amount of an anti-pathogenic agent
to impair growth and/or viability of the target pathogenic organism. A
formulation containing the antipathogenic agent is introduced to a plant
tissue or part. For example, the formulation is sprayed on, as a wet or
dry formulation, the surface and/or underside of the leaves or other plant
tissue or part of a plant infected with a plant pathogen, or of a plant
susceptible to infestation with a plant pathogen, preferably to the point
of run off when a wet formulation is used a plant growth promotant, such
as saponin, is optionally used either in the antipathogenic formulation or
as a separate formulation. Alternately, the formulation can be applied wet
or dry to the rhizosphere where it can contact the roots and associated
pathogenic organisms which colonize the roots. In some instances,
time-release formulations may find use, particularly for applications to
the rhizosphere.
The method of introducing into the target organism can be by direct
ingestion by the pathogenic organism, for example, an insect or a fungus,
from a treated plant surface, or by feeding of a pathogenic organism on a
nutrient-providing surface of a host entity which is colonized by the
target pathogenic organism which either contains or has on its surface the
antipathogenic agent. The presence of the anti-pathogenic agent on a
nutrient-providing surface of a host plant can be a result of direct
contact of the anti-pathogenic agent with the plant part or it can be by
elaboration from the host plant as a result of induction of systemic
resistance as a secondary effect to prior treatment of the plant with the
anti-pathogenic agent, or as a result of genetic modification of the host
plant.
A preferred method for producing a desired component of the present
formulations in a plant host is through recombinant DNA means.
Particularly by modifying the level of at least one compound of interest
of the formula (1), (2), (3), (4) and/or saponin in plant tissues of
interest through construction of transgenic plants using recombinant
techniques known in the art. The methods involve transforming a plant cell
of interest with an expression cassette functional in a plant cell
comprising as operably linked components in the 5' to 3' direction of
transcription, a transcriptional and translational initiation regulatory
region, joined in reading frame 5' to a DNA sequence encoding and capable
of modulating the production and/or required to produce the compound of
interest, and translational and transcriptional termination regions.
Expression of an enzyme required to produce the compound of interest
provides for an increase in production of the compound as a result of
altered concentrations of the enzymes involved in the compounds'
biosynthesis.
One or more compounds of the present formulations can be introduced to the
target organism by modulating the expression of one or more genes a gene
encoding or more enzymes or an enzyme pathway or cluster required to
control the level of the compound of interest in a plant, plant part,
plant cell, specific plant tissue and/or associated with a particular
stage of plant growth. The enzyme or enzymes can be in a biosynthetic
pathway or a degradation pathway and the regulation will be up or down
respectively; i.e., to modulate expression of an indigenous or an
endogenous plant gene an indigenous plant gene is one which is native to
the genome of the host plant. An endogenous plant gene is one that is
present in the genome of the plant host of interest, and may be an
indigenous gene or a gene that is present as a result of infection of the
plant A, a viral gene), or otherwise naturally incorporated into the plant
genome. The host plant also can be modified by recombinant means or by
traditional plant breeding methods to introduce one or more genes
exogenous to the host plant which encode enzymes which control the level
of the compound of interest and/or are in the synthetic pathway for one or
more compounds of formula (1), (2), (3) or (4) and/or saponin. By
"modulation of gene expression" it is intended control of production of a
gene product of interest at the level of transcription, translation and/or
post translation. The level of the compound of interest is controlled by
modulating the expression of one or more endogenous genes or transgenes
encoding one or more enzymes required to synthesize the compound of
interest.
Methods for modulating gene expression in plants are known in the art.
Variation in growth conditions or exogenous application of compounds to a
plant can affect gene expression. For example, the formulations of the
present invention can be used to induce systemic plant resistance through
modulation of endogenous gene expression. At the molecular level, gene
expression depends substantially on the transcription, translation and
termination control regions which regulate expression of a structural gene
coding region. By exploiting the plant signals which regulate these
control regions or by the direct recombinant manipulation of the control
regions, expression of a gene encoding an enzyme required to control the
level of cinnamic aldehyde, for example, can be modulated. For use in a
transgene supplied exogenously to a plant host, the transgene will include
control regions that are selected and designed to achieve the desired
level and timing of gene expression. As appropriate, the control regions
may be homologous (native) or non-homologous (non-native) to the gene of
interest. By "homologous" it is meant that the control region(s) is from
or substantially similar to a control region normally associated with the
gene of interest. By "non-homologous" it is meant that the control
region(s) originates from a different nucleotide source or sequence or is
substantially different from the control region(s) normally associated
with the gene of interest. For example, if the enzyme coding sequence is
non-homologous in source as compared to the control regions, in order to
have expression of the gene in a plant cell of interest, transcriptional
and translational initiation regulatory regions or promoters functional in
these plant cells must be provided operably linked to the coding sequence.
Transcription and translation initiation signals functional in plant cells
include those from genes which are present in the plant host or other
plant species, and direct constitutive or selective expression in a plant
host.
DNA constructs for expressing a gene of interest are prepared which provide
for integration of the expression cassette into the genome of a plant
host. Integration can be accomplished using transformation systems known
in the art such as Agrobacterium, electroporation or high-velocity
microparticle-mediated transformation. For biosynthesis of cinnamic and/or
coniferyl aldehyde, plant cells are transformed with an expression
cassette comprising DNA encoding a structural gene for one or more enzymes
required to synthesize cinnamic and/or coniferyl aldehyde and capable of
increasing the amount of these aldehydes in the tissue of interest. Of
particular interest are those genes encoding one or more enzymes capable
of metabolizing a precursor compound required for the biosynthesis of the
saponin, cinnamic and/or coniferyl aldehyde compound of interest from
substrates normally found in a plant cell. Similarly, for saponin
biosynthesis, plant cells are transformed with an expression cassette
comprising DNA encoding a structural gene for one or more Ames required to
synthesize saponin.
Depending upon the application, cinnamic aldehyde, saponin or one of the
other compounds of interest can be preferentially expressed in a tissue of
interest and/or a particular organelle. Of particular interest is the
selective control of cinnamic and/or coniferyl aldehyde production and/or
saponin in plant tissues such as leaves, roots, fruits and seeds; the
tissue site can be varied depending upon the site of infestation of the
target pathogen. Tissue specificity is accomplished by the use of
transcriptional regulatory regions having the desired expression profile.
Translocation of the enzyme to a particular organelle is accomplished by
the use of an appropriate translocation peptide. Methods for tissue and
organelle specific expression of DNA constructs have been described are
known in the art ([refs]). For example, promoters showing differential
expression patterns in fruit are described in U.S. Pat. No. 4,943,674 and
U.S. Pat. No. 5,175,095; in seed in U.S. Pat. No. 5,315,001; in rapidly
developing tissues and tender shoots in U.S. Pat. No. 5,177,011.
To verify regulation and expression of the gene of interest, various
techniques exist for determining whether the desired DNA sequences present
in the plant cell are integrated into the genome and are being
transcribed. Techniques such as the Northern blot can be employed for
detecting messenger RNA which codes for the desired enzyme. Expression can
further be detected by assaying for enzyme activity or immunoassay for the
protein product. Most preferably the level of the compound of interest
present in a plant host is measured using methods known in the art
([refs]). A desired phenotype, for example, is increased cinnamic aldehyde
content in a plant tissue of interest as measured by expression of the
gene of interest and/or the level of cinnamic aldehyde present in the
plant host as compared to a control (non-transgenic) plant.
For introduction of one or more compounds of the present formulations to
the target organism, a plant host expressing a gene encoding an enzyme
required to control the level of the compound of interest results in the
exposure of a target organism to at least one component of the
antipathogenic formulation. In another embodiment, selective expression of
the gene of interest provides for systemic plant host resistance to
pathogen attack or colonization. At least one component of the
antipathogenic formation can be expressed by the transgenic plant host and
optionally other components of the antipathetic formulation are
exogenously applied to the plant host so that the combination elicits the
desired antipathogenic effect when either directly or indirectly
introduced to the target organism.
Transgenic plants having an increased ability to accumulate aromatic
aldehydes such as cinnamaldehyde and coniferyl aldehyde to provide
self-protection against plant pathogens or be used as a natural source of
aromatic aldehydes for extraction and subsequent use as a chemical
pesticide can be prepared.
Accumulation of aromatic aldehydes can be achieved by downregulating the
expression of specific plant genes that encode enzymes which either cause
further metabolism of the desired aldehydes or divert metabolic
intermediates away from the desired aldehydes. In the case of
cinnamaldehyde, for example, this involves downregulating the expression
of cinnamate 4-hydroxylase (CA4H) and cinnamic alcohol dehydrogenase
(CAD). By reference to FIG. 7, it can be seen that CA4H ordinarily diverts
some cinnamic acid away from cinnamaldehyde to produce p-coumaric acid,
itself a metabolic intermediate. Reducing CA4H activity alone is not
sufficient to cause accumulation of cinnamaldehyde because CAD can rapidly
convert cinnamaldehyde to cinnamyl alcohol, which then becomes
incorporated into lignin or accumulates as glycosides. Simultaneously
reducing both CA4H and CAD activities results in increased metabolic flux
from cinnamic acid into cinnamaldehyde and decreased conversion of
cinnamaldehyde into cinnamyl alcohol. Some cinnamaldehyde becomes
incorporated into lignin but cinnamaldehyde (either free or as glycosides)
also accumulates to above-normal levels, particularly at times when the
biosynthesis of cinnamic acid is elevated. This occurs when the level of
phenylalanine ammonia lyase (PAL; the first and rate-limiting step in
general phenylpropanoid metabolism, Hahlbrock and Scheel (1989) Annu. Rev.
Plant Physiol. Plant Mol. Biol. 40:347-369) activity is high, a situation
that naturally occurs in plants in response to a wide range of stimuli
including invasion by fungal pathogens and mechanical damage associated
with wounding and insect feeding.
Inhibiting CAD activity in transgenic plants has been proposed as a method
of reducing lignin synthesis in plants and thereby improving the
digestibility of fodder crops (WO 93/05159). These experiments suggested
that lignin biosynthesis had been altered qualitatively, but not
necessarily quantitatively, but did not demonstrate or appreciate the
desirability of accumulating cinnamaldehyde as a method of increasing
protection against pathogens.
A number of plant CA4H and CAD genes have been cloned and their sequences
are available from GenBank. Portions of these genes that include
nucleotide sequences that are conserved between different plant species
can be used directly in a plant expression vector (antisense or sense
orientation) to suppress the expression of the corresponding endogenous
genes (e.g., Pear, et al., The Plant Cell Antisense Res. and Develop.
(1993) 3:181-190, Napoli, et al., The Plant Cell (1990) 2:279-289. More
preferably, these conserved gene sequences are used to isolate CA4H and
CAD cDNA clones from a cDNA library of the plant species that is to be
modified. The resulting cDNA clones, or portions thereof, are then
introduced into a plant expression vector (antisense or sense) and used to
transform the plant(s) of interest. DNA constructs according to the
invention preferably comprise a sequence of at least 50 bases which is
homologous to the endogenous CA4H or CAD genes.
A recombinant DNA molecule can be produced by operatively linking a vector
to a useful DNA segment to form a plasmid that can be used for plant
transformation. A vector capable of directing the expression of RNA from a
cloned portion of a gene is referred to herein as an "expression vector."
Such expression vectors contain expression control elements including a
promoter. Typical vectors useful for expression of genes in higher plants
are well known in the art and include vectors derived from the Ti plasmid
of Agrobacterium tumefaciens described by Rogers et al., Methods in
Enzymology (1987) 153:253-277. A common promoter that is used to provide
strong constitutive expression of an introduced gene is the cauliflower
mosaic virus (CaMV) 35 S promoter (available from Pharmacia, Piscataway,
N.J.). Either constitutive promoters (such as CaMV 35S) or inducible or
developmentally regulated promoters (such as the promoter from a PAL gene
or the endogenous CA4H or CAD genes) can be used. Use of a constitutive
promoter will tend to affect functions in all parts of the plant, while
use of an inducible or developmentally regulated promoter has the
advantage that the antisense or sense RNA is only produced in the tissue
and under the conditions it is required. The use of developmentally
regulated promoters is preferred in the use of this invention because the
down-regulation of phenylpropanoid biosynthesis is known to be capable of
producing undesirable side-effects on the development of transgenic plants
containing a heterologous PAL gene (Elkind, Y. et al., 1990) Proc. Nat.
Acad. Sci. (1990) 87:9057-9061.
A number of different transformation methods are available for the routine
transformation of a wide range of plant species. One method that is
particularly efficient for the transfer of DNA into dicotyledonous plants
involves the use of Agrobacterium. In this method the gene of interest is
inserted between the borders of the T-DNA region that have been spliced
into a small recombinant plasmid with a selectable marker gene (for
example encoding neomycin phosphotransferase II or phosphinothricin
acetyltransferase). The recombinant plasmid is then introduced into an
Agrobacterium host by transformation or triparental mating. The
Agrobacterium strain carrying the gene(s) of interest is then used to
transform plant tissue by co-culturing the bacteria with an appropriate
plant tissue (e.g., leaf disc). Transformed cells are selected in tissue
culture using the appropriate selection agent and plants are then
regenerated (see Horsch, R. B. et al., Science (1985) 227:1229-1231. Other
methods that have been used in the transformation of plant cells, and in
particular the more recalcitrant crop plants, include biolistics and
electroporation (for detailed protocols, see Sanford, et al., (1993)
Methods in Enzymology 217:483-509; and Potter, (1993) Methods in
Enzymology 217:461-478.
Once transgenic plants have been produced, conventional enzyme assays for
CA4H and CAD are used to determine the level of suppression of enzyme
activity achieved in different transformants. It is likely that only a
small fraction of the transformants produced will have a sufficiently low
residual enzyme activity to cause the accumulation of aromatic aldehydes
without also producing some undesirable side-effects on plant development.
For this reason, a preferred method of producing the desired transformants
with both CA4H and CAD suppressed is to introduce the two genes separately
into different transformants and then combine them by standard sexual
crosses. This permits a larger number of combinations of level of gene
suppression to be evaluated at the same time.
An alternative to overproducing aromatic aldehydes in transgenic plants is
to use the plant genes to confer on a microbial host the capability of
synthesizing specific aromatic aldehydes. The resulting microbes may be
used either to produce the flavonoid aldehydes in a fermentation system or
as a natural delivery system of the aromatic aldehydes in viable or
non-viable microbial preparations. Yeasts, especially Saachoromyces
cerevisiae, are preferred organisms for this purpose because they have
already been engineered for high-level expression of PAL (Faulkener, J. D.
B. et al., Gene 143:13020, 1994) and a plant cinnamate 4-hydroxylase has
been shown to function in yeast (Urban, et al. 1994 Eur. J. Biochem
222:843-850.
The expression of PAL introduces the capability to produce cinammic acid
from phenylalanine. Two additional enzymic steps are required to produce
cinnamaldehyde from phenylalanine. In plants, these steps are catalyzed by
the enzymes cinnamate:CoA ligasc (CL) and cinnamoylCoA reductase (CCoAR)
but as 4-coumarateCoA ligase (4CL) can also use cinnamic acid as substance
(Knobloch, and Hahlbrock 1977, Arch. Biochem. Biophys. 184:237-248, 4Cl
can be used instead of CL. More than 20 cloned PAL genes and more than 6
4CL genes have been described in sufficient detail (GenBank) to facilitate
their use in practicing the current invention. A gene for a CCoAR is
obtained by applying standard gene cloning techniques to isolate a cDNA
clone using as a probe sequence derived from the amino acid sequence of
the N-terminus, or peptide fragments, of the purified protein. CCoAR has
been purified and partially characterized from soybean cultures
(Wengenmayer et al., (1976) Eur. J. Biochem, 65:529-536; Luderitz, and
Grisebach, Eur. J. Biochem, 119:115-124, 1981), spruce cambial sap
(Luderitz, and Grisebach, supra), poplar xylem (Sarni, et al., Eur. J.
Biochem, 139:259-265, 1984) and differentiating xylem of Eucalyptus gunnii
(Goffner, et al., Plant Physiol. 106:625-632, 1994). The preferred method
of purification is that of Goffner et al. (supra) because it results in a
single protein band on SDS-polyacrylamide gels that an be used for protein
sequencing.
The cloned genes are introduced into standard expression vectors and used
to transform a microbial host, preferably yeast, by standard
transformation techniques such as electroporation (Becker, and Guarante,
Methods in Enymol, 194:182-187, 1991). Standard enzyme assays are used to
confirm the functional expression of the engineered genes and assays for
aromatic aldehydes are used to select stains with maximal production.
Because aromatic aldehydes have antimicrobial properties it is preferred
to use expression vectors that will cause expression of the introduced
genes only late in the growth cycle or in response to a chemical inducer.
It may also be desirable to grow the engineered microbia host in an
immobilized whole cell reactor (e.g., Evans, et al., Biotechnology and
Bioengineering 30:1067-1072, 1987) to prevent the aldehydes from
accumulating in the culture medium.
The target pathogenic organisms include fungi which colonize a surface of a
part of a plant which is an elicitor for the fungus. By elicitor is
intended that the plant secretes nutrients required by the fungus.
Examples of fungi and the plant parts which the colonize are as follows.
Black spot on fruit; Fusarium sp. on flowers roots and leaves; and
Fusarium spp. and aspergillus on roots and leaves. Fusarium causes
vascular wilts of annual vegetables and flowers, herbaceous perennial
ornamentals, plantation crops and the mimosa tree. Different plants are
attacked by special forms or races of the fungus. Verticulum (V.
albo-atrium and V. dahlise) cause vascular wilts and colonize roots,
flowers and leaves. In addition the following also constitute target
organisms: Phragmidium spt; Diplocaopan rosae; Sphaerotheca tannosa;
Oibiapsis sicula; Phytophoya taraesitica; Puccinia spp; Alternaria sp;
Susaiun spp; Botrytis cinera; Sclerotinia Homoeocarca; Dutch Elm disease
(Ceratocystis ulmi) and oak wilt (C. fagacearum). Ceratocystis causes
vascular wilts, mainly of trees.
Target organisms also include insects, particularly those of the orders
Orthoptera; Thysanoptera which includes hips; and Homoptera which include
aphids, leafhoppers, white flies, mealy bugs, cicadas and scale insects.
It is a theory of the invention that the insects which are susceptible to
treatment with the subject formulations are those which harbor symbiotic
bacteria in their gut. Accordingly, insects other than those listed which
harbor symbiotic material also can be controlled with the subject
formulations. Other target organisms include arachnids, particularly
spider mites (arthropoda).
Plants which are of interest for treatment are those which are colonized by
pathogenic organisms and include flowering plants, grasses, including bent
grass, vegetables, cereals and fruits including tomato, potato, artichoke,
strawberries, corn, cereal grains, onion, cucumber, lettuce, tobacco, and
citrus such as orange, lemons, limes and grapefruit, as well as bell peers
and grapes, and fruit trees such as peach, apple and cherry, ornamentals
such as roses and trees, particularly conifers. Also included are crops
intended for consumption by fish, fowl and animals, including humans,
directly or indirectly. By "directly or indirectly" is intended that the
crops could be ingested, for example, by humans (direct consumption), or
that it is the nonhuman animal or fowl which ingests the crop and is in
turn ingested by humans (indirect consumption). Crops intended for
consumption include tobacco, fish, animal and fowl fodder, crops intended
for processing into alcohol or food products such as corn syrup, and the
like.
Of particular interest is treatment of plants affected by powdery mildew
which is caused by target organs which are species of fungi of the family
Erysiphaceae. Generally the genera are distinguished from each other by
the number (one as opposed to several) of asci per cleistotheciun and by
the morphology of hypal appendages growing out of the walls of the
creistothecium. As an example the following genera cause powdery mildew in
the indicated plants: Erysiphe cichoracearum, begonia, chrysanthemum,
cosmos, cucurbits, dahlia, flax, lettuce and zinnia; E. graminis, with
cereals and grasses; E. polgoni, beans, soybeans, clovers, and other
legumes, beets, cabbage and other crucifers, cucumber and cantaloupe,
delphinium and hydrangea; Microsphaera alni, blueberry, catalpa, elm,
lilac, oak, rhododendron, and sweet pea; Phyllactinia sp. catalpa, elm,
maple and oak; Podosphaera leucotricha, apple, pear and quince; P.
oxyacanthae, apricot, cherry, peach and plum; Spaelrotheca macularis,
strawberies; S. mors-uvae, gooseberry and currant; S. pannosa, peach and
rose; and Uncinula necator, grape, horse chestnut and linden.
Also of particular interest is the treatment of plants affected by rust
caused by Basidiomycetes of the order Uredinales. These plant rusts are
among the most destructive of plant diseases. They have caused famines and
ruined the economics of large areas, including entire countries. There are
about 4,000 species of rust fungi. The most important rust fungi and the
diseases they cause follow: Puccinia, causing severe and often
catastrophic diseases on numerous hosts such as the stem rust of wheat and
all other small grains (P. graminis); yellow or stripe rust of wheat,
barley and rye (P. striiformis); leaf or brown rust of wheat and rye (P.
recondita); leaf or brown drarf rust of barley (P. hordei); crown rust of
oats (P. coronata); corn rust (P. sorghi); southern or tropical corn rust
(P. polysora) sorghum rust (P. purpurea); and sugarcane rusts (P. sacchari
and P. kuehnii).
Puccinia also causes severe rust diseases on field crops such as cotton (P.
stakmanii); vegetables such as asparagus (P. asparagi); and flowers such
as chrysanthemum (P. chrysanthemi), hollyhock (P. malvacearum), and
snapdragon (P. antirrhini). Gymnosporangium, causes the important
cedar-apple rust (G. juniperi-virginianae) and hawthorn-cedar rust (G.
globosum). Hemileia, causes the devastating coffee leaf rust. (H.
vastatrix). Phragmidium, causes rust on roses and yellow rust on
raspberry.
Uromyces: several species cause the rusts of legumes (bean, broad bean, and
pea) and one causing rust of carnation (U. caryophyllinus). Cronartium,
causes several severe rusts of pines, oaks, and other hosts, such as the
white pine blister rust (C. ribicola); fusiform rust of pines and oaks (C.
quercuum f. sp. fusiforme); eastern gall or pined rust (C. quercuum f. sp.
virginianae); pine-sweet fern blister rust (C. comptoniae); pine-Comandra
rust (C. comandrae); and southern cone rust (C. strobilinum). Melampsora,
causes rust of flax (M. lini). Coleosporium, causes blister rust of pine
needles (C. asterinum). Gymnoconia, causes orange rust of blackberry and
raspberry. Phakopsora, causes the potentially catastrophic soybean rust
(P. pahyrhizi). Tranzschelia, causes rust of peach.
For treatment of powdery mildew, rust and other pathogens which colonize
the leaves of the host plant, the host plants are sprayed to run off with
a formulation of the invention. The amount of compound(s) of formula (1)
used will vary depending in part upon the target pathogen and the host
plant and can be determined empirically by evaluating the sensitivity of
the target organism to the formulation and the phytotoxic effects of that
formulation or the host plant. The plants can be sprayed prior to or after
infestation, preferably prior to infestation. However, in order to
minimize damage to the host plant, where feasible, it is preferable to
treat older plants, as young green leaves tend to be more susceptible to
phytotoxicity. Alternatively, transgenic crops can be used, which express
one or more components of the formulation in an amount sufficient to
inhibit growth of the pathogen and/or kill the pathogen. Preferably the
component(s) is expressed in the tissue colonized by the pathogen, for
example the leaves.
Also of particular interest is treatment of phylloxera infestation in
grapes. For this application, it is necessary to deliver the formulation
to the roots of the plant to the location of the insect colony. Typically,
phylloxera are found as deep as the roots of the host plant, which may be
eight feet or deeper. When used in a solid form or microencapsulated, the
dosage used is typically on the order of 1% to 35% on a w/w basis, the
maximum loading to be determined as a function of shell material selected.
Analytical chemical techniques are used to determine and optimize rate of
release. For qualitative purposes GC techniques can be used to determine
the amount of aldehyde released. The samples of encapsulated (pelletized)
product are mixed with the soil types selected and sampled at different
time periods to measure release. Alternatively, volatile gases released
from the formulation can also be analyzed. For measuring the activity of
foliar and drip irrigation applications the stability of the formulations
over time can also be evaluated using the GC methodology using methods
known to those skilled in the art. Methanol or alcohol extractions of the
formulations also can be prepared for HPLC analysis. The aldehyde
components can be coupled to a solid support, optionally through a linker
such as a polysaccharidase binding domain, where the solid support is a
polysaccharide such as cellulose, particularly microcrystaline cellulose.
The preparation of cellulose binding domains is described in U.S. patent
application Ser. Nos. 5,340,731; 5,202,247 and 5,166,317. The aldehydes
can be coupled to the binding domains, with or without a cleavable bond,
using methods well known to those skilled in the art. As in the case of
treatment of powdery mildew and rust, transgenic crops can be used; for
treatment of phylloxera the preferred tissue of expression of components
of formula (1) is the root.
In addition to treating a host plant, seeds can also be treated using the
subject formulations. The seeds can be dusted with a powder preparation
(see U.S. patent application Ser. No. 4,978,686 for examples of inorganic
materials to which the formulations can be adsorbed) or admixed in a plant
substrate such as vermiculite. Seeds also can be obtained from transgenic
crops, wherein the components of formula (1) have been expressed in seed,
preferably preferentially in seed. Seedlings grown under sterile
conditions from treated seeds are free of susceptible fungi and insects.
Additionally, seedlings also can be treated with the subject formulations.
In some instances it may be necessary to adjust the treatment formulation
so as to reduce any phytotoxicity associated with the treatment as tender
young shoots are more likely to exhibit phytotoxicity symptoms.
In order to determine the susceptibility of particular fungi or insects to
the claimed compositions, in vitro and in vivo tests ]mown to those
skilled in the art are used. The mean disease control can be calculated
for particular pathogens on particular host plants. Generally it is
desirable to obtain a mean disease resistance of 60% or better, preferably
at least about 70%. The formulations also need to be evaluated for
phytotoxicity; it therefore is important that at least one evaluation of
the toxicity of the formulations be on living plants of the host variety.
Phytotoxicity can be rated as follows in order of increasing severity of
toxicity: 0-plants without any symptoms; 1-very slightly browning of
hypocotyl (no other symptoms); 2-some wilting of plant, dying of lower
leaves, some browning of vascular system; 3-wilting of entire plant,
leaves dying, hypocotyl with external and internal symptoms; 4-necrosis of
stem, plant dying. It is preferable that the formulation used have a
phytotoxicity rating of 2 or less, more preferably 1 or less.
The components of a formulation to be used for a particular application can
be determined by constructing a dose response curve by evaluating first
the concentration range over which a given component has no activity to
where it provides maximum activity and then evaluating this component
separately and in combination the components of interest for a given
formulation. As an example, the effects of cinnamic aldehyde in a range
from 0.1 ppm to 25,000 ppm on powdery mildew is evaluated. At a dose of
0.05%, it provides 3% mean disease control. The mean disease control can
be increased by using higher doses of cinnamic aldehyde, and/or adding
other compounds of formula (1), or by increasing the substantiveness of
the formulation by adding detergent, and the like. The antipathogenic
and/or phytotoxic effect of a particular formulation on a given pathogen
and/or plant host is then measured for each formula and component with or
without a serial dilutent of any additional component of interest. Optimal
dose-ranges are calculated in vitro and in vivo using techniques known to
those of ordinary skill in the art. Preferred formulation provide: a mean
disease resistance of at least about 60% or better with an optimum of
about 70% or greater; and/or a phytotoxicity rating of 2 or less, with 1
or less being optimum.
The following examples are offered by way of illustration and not by way of
limitation.
EXAMPLES
Materials and Methods
The chemicals used in the examples given below were obtained from the
following sources: cinnamic aldehyde, Spectrum Chemical Company, N.J.;
coniferyl aldehyde, APIN Chemical, U.K.; Tween 80 and sodium bicarbonate
Spectrum Chemical Company, Gardena, Calif.
Example 1
Treatment of Powdery Mildew on Rose Cultivars
Potted Roses. Eight cultivars of rose were to investigate the effect of a
cinnamic aldehyde/NaHCO.sub.3 formulation on rust and spores. The
cultivars used included Moss unnamed (Moss), Galica, Rosette Delize
(Hybrid Tea), Rosa Rugosa Rubra (Rugosa), Abel Morrison (Hybrid
perpetual), John Laing, Betty Prior, and Rose de Roi. Five (5) potted
cultivars (Moss, Galica, Hybrid Tea, Rugosa, and Hybrid perpetual) were
selected and assigned a disease rating after Paulus and Nelson (supra) for
powdery mildew, rust and spores..sup.1 The Moss and Galica cultivars were
5 on a scale of 0-5 (where 0=no powdery mildew rust/spores lesions,
1=1-25, 2=26-50, 3=51-75, 4=76-90, and 5=>90% total leaves per bush). The
Hybrid Tea and Hybrid perpetual were rated 3 and the Rugosa was rated 1.
The Moss and Galica also were infected with rust equivalent to a 3 rating.
1. Spores were evaluated only on Moss, Galica and the control plants.
Each cultivar received a foliar spray of about 100 ml of a cinnamic
aldehyde formula containing 5 g cinnamic aldehyde, 80 g NaHCO.sub.3, 10 g
of Tween 80 and water to 1000 g. In addition, 250 ml of 0.01% (v/v)
aqueous solution of 10.degree. brix saponin extract from the yucca
shidigera plant was administered to each potted plant once a week
beginning with the date of the fist cinnamic aldehyde/NaHCO.sub.3
treatment. Control plants received no treatment. A single treatment was
eradicative of powdery mildew, rust, and spores though the final weekly
field observation eight weeks later as compared to the no treatment
controls which remained at disease ratings of 5, 3, and 4 for powdery
mildew, rust and spores, lively. Moreover, the treatment appeared to have
induced systemic resistance. No phytoxicity was observed.
Field grown roses. Another experiment was designed for field grown cut
flower rose to evaluate the efficacy of powdery mildew control by cinnamic
aldehyde/NaHCO.sub.3 during the same period (season) and environmental
conditions. Powdery mildew and rust inoculum were high in the test field,
and no additional inoculum was necessary to provide disease pressure.
Cultivars John Laing, Betty Prior, and Rose de Roi were used in tis
investigation. Eight John Laing plants from a block row of sixteen were
selected for treatment. Every other plant beginning with the first plant
in the row was treated. Three Betty Prior plants were selected from a
block of six were similarly treated, as were two Rose de Roi plants from a
block of four. A single foliar spray treatment (about 100 ml) of a
cinnamic aldehyde 5 g, and Tween 80, 10 g and NaHCO.sub.3 80 g and water
to 1000 g was applied to each setting of cultivars. Plants were an average
of 0.86 m apart. The disease rating was the same as that used to evaluate
powdery mildew in containerized cultivars. Controls were untreated plants.
Absence of wind and exact spraying protected controls from spray drift.
The John Laing cultivars were young, 45day-old plants with a rating of 5
for powdery mildew. The Betty Prior cultivars were older (>240 days),
previously sprayed with Eagle (120 days prior) with a rating of 3 for
powdery mildew and the Rose de Roi were 240days-old plants with a rating
of 2 for powdery mildew and 2 for rust using the same scale as provided
above. Induced systemic resistance was determined by observing the number
of lesions of powdery mildew and rust produced on each plant after
treatment as compared to untreated controls. Weekly reviews were made of
the various plants. The effect on growth increase of the treatment regimen
was determined at the last field observation of each plant.
With the exception of the untreated controls and three plants of cultivar
Betty Prior which had reinfection of powdery mildew with a rating of 3,
all plants were free of powdery mildew at the end of the five week trial.
No phytotoxicity was observed. All plants had new growth exceeding that of
the untreated controls.
The Mean Percentage of Disease Control (MPDC) was calculated for even group
of plants. The results were as follows for powdery mildew: John Laing,
98.3%; Betty Prior, 64.3%; Rose de Roi, 100%. The average for all three
roses was 90.7% for powdery mildew. Rust was evaluated only on Rose de
Roi, and was 85.0%. Effective fungicides for powdery mildew should provide
a MPDC of .gtoreq.70% under Greenhouse or field conditions, and for rust
.gtoreq.65%.
Example 2
Treatment of Fungi and Insect on Roses with Coniferyl Aldehyde
Six cultivars of infected rose in dedicated experimental rose gardens were
used. Four of Mrs. John Laing (Hybrid perpetual) and two of Marchionese of
Londonderry (Hybrid perpetual) were treated with one of two formulations
of coniferyl aldehyde. The low dose treatment (T1) was a coniferyl
aldehyde, 10 g of Tween 80, 80 g of NaHCO.sub.3 and 905 g of H.sub.2 O for
1000 g of product. The high dose treatment (12) was a coniferyl aldehyde
formula comprising of 100 g of coniferyl aldehyde, 20 g Tween 80, 120 g
NaHCO.sub.3, 760 g H.sub.2 O for 1000 g of product. See Table 1.
The first two Mrs. John Laing plants (P1 and P2) were assigned a disease
rating of 3 for powdery mildew and rust after Paulus and Nelson (supra).
Mrs. John Laing plants 3 and 4 (P3 and P4) were assigned a disease rating
of 4 and 5 respectively for powdery mildew and rust. P3 and P4 also were
infected with aphids, each plant with >35 insects. Both Marchionese of
Londonderry (P5 and P6) were rated 5 for powdery mildew and rust after
Paulus and Nelson (supra). Two treatment formulae were used for this
screen trial. Each plant (P1 through P6) received a .apprxeq.100 ml
treatment spray of as shown in the Table 1 below. Control plants received
no treatment (i.e. they were sprayed with water alone). The change in the
rating (PRE-POST) was calculated as the mean percentage of disease control
(MPDC). MPDC is defined by the formula:
MPDC is defined by the formula:
##EQU1##
and
MDIC=Mean % of disease incidence in untreated controls
MDIT=Mean % of disease incidence in the treatment
TABLE 1
Plant - Treatment/Dose Assignment
Treatment/Dose Plant
T1 - Low P1, P4, P6
T2 - High P2, P3, P5
As shown in Table 2 below, both formulas reduced (pre-post treatment
change) levels of infection. Both powdery mildew and rust levels of
infection were reduced a minimum of one rating category after treatment as
compared to plants sprayed with water alone.
TABLE 2
Plant
Treatment/Dose
Low (T1) High (T2)
P1 P4 P6 P2 P3 P5
PEST
Powdery Mildew Pre 3 3 4 5 5 5
Post 2 1 1 2 1 1
Change 1 2 3 3 4 4
Rust Pre CFU 3 3 4 5 5 5
Post CFU 2 1 3 2 1 1
Change 1 2 1 3 4 4
Aphids Pre # -- 35 -- .gtoreq.35 --
Post # -- 0 -- -- --
Change -- .gtoreq.35 -- .gtoreq.35 --
Aphids were eliminated from P3 and P4 indicating that the formulas have
insecticidal properties. Coniferyl aldehyde, as is cinnamic aldehyde,
shared antibiotic properties and may eliminate symbiotic bacteria present
in the host insect without which the insect cannot live.
Treatment of Powdery Mildew on Rose
A three treatment experiment with Cinnamic Aldehyde Formula and Components,
Coniferyl aldehyde formula and combined Cinnamic and Coniferyl aldehyde
formula was evaluated on field grown roses known to be susceptible to
powdery mildew. The plants were blocked by variety before fungicide
treatments and were randomized as to the plants. Two varieties were used
in each of the three experiments described below. In experiment 1,
Reichsprasident von Hindenburg (Bourbon) and Oskar Cordel (Hyvrid
Perpetual) were used; in experiment 2, Rosa Gallica Officinalis
(Apothecary Rose) and Deuil de Paul Fontaine (hybrid Moss) were used. In
experiment 3 Comte de Chambord (Portland) and Madame Pierre Oger (Bourbon)
were used. Experiment 1 evaluated the effect of cinnamic aldehyde alone
and in combination with Tween 80 and/or NaHCO.sub.3 components, experiment
2 evaluated the effect of Coniferyl aldehyde, and experiment 3 evaluated a
combination of cinnamic aldehyde and coniferyl aldehyde with Tween 80
and/or NaHCO.sub.3. Nine treatments were tested in experiment 1, six in
experiment 2 and six in experiment 3. See Table 3 for treatment protocol;
formula 1 was used for these experiments.
Each plant received a single foliar spray of 100 ml following evaluation of
powdery mildew infection (after Paulus and Nelson). The response variable
recorded for each plant was the powdery mildew infection rating based on
the Paulus/Nelson rating scale. Plants were evaluated on this scale just
prior to and four days after treatment. Mean percentage of disease control
data indicate that all three combination formulas (i.e. G, M, and Q
provided in excess of 70% disease control based on these experiments. See
Table 4. Treatment Q was significantly better than all other treatments,
including benomyl. Moreover, cinnamic aldehyde, coniferyl aldehyde, Tween
80 and NaHCO.sub.3 are used in the food industry and there is likely to be
little toxicological risk to the consumer or handler from any
horticultural or food crop sprayed in this way. Similarly, as these
chemicals leave no toxic residue, there is little chance of any
detrimental effect on the wider environment, and their use is likely to be
compatible with current biological control methods.
TABLE 3
Treatment Protocol
Amount of treatment
Treat- ingredient(s).sup.1
Group ment Ingredient(s) Formula 1 Formula 2
1 A Cinnamic aldehyde 5 g 20 g
(CNMA)
1 B Tween 80 (T80) 10 g 20 g
1 C NaHCO.sub.3 80 g 60 g
1 D CNMA + T80 5 g, 10 g 20 g, 60 g
1 E CNMA + NaHCO.sub.2 5 g, 80 g 20 g, 60 g
1 F NaHCO.sub.3 + T80 80 g, 10 g 60 g, 20 g
1 G Formula 1 (CNMA) A = 5, A = 20,
B = 10 g, B = 20,
C = 80 g C = 60 g
1,2,3 H +Control.sup.2 per R,S,T.sup.2
manufacture
instructions
1,2,3 I -Control H.sub.2 O H.sub.2 O
2 J Coniferyl aldehyde 5 g 20 g
(COFA)
2 K COFA + T80 5 g, 10 g 20 g, 20 g
2 L COFA + NaHCO.sub.3 5 g, 80 g 20 g, 60 g
2 M Formula 2 (COFA) J = 5 g, J = 20 g,
B = 10 g, B = 20 g,
C = 80 g C = 60 g
3 N CNMA + COFA 2.5 g, 2.5 g 10 g, 10 g
3 O CNMA + COFA + T80 2.5 g, 2.5 g, 10 g, 10 g,
10 g 10 g
3 P CNMA + COFA + NaHco.sub.3 2.5 g, 2.5 g, 10 g, 10 g,
80 g 60 g
3 Q Formula 3 (CNMA + A = 2.5 g, A = 10 g,
COFA) J = 2.5 g, J = 10 g,
B = 10 g, B = 20 g,
C = 80 g C = 60 g
.sup.1 Balance H.sub.2 O to 1000 g
.sup.2 R = Benomyl
.sup. S = Malathion
.sup. T = Lilly Miller SSKB
TABLE 4
Effect of Cinnamic Aldehyde and Coniferyl Aldehyde
Formulations on Rose Powdery Mildew
Aldehyde
Cinnamic
Cinnamic Coniferyl Aldehyde (2.5 g) +
Additive Aldehyde Aldehyde Coniferyl
Formulation None (5 g) (5 g) Aldehyde (2.5 g)
Mean % Disease Control
None 0% 50% 56% 69%
T80 (10 g) 0% 44% 44% 69%
NaHCO.sub.3 44% 56% 44% 88%
T80 + NaHCO.sub.3 19% 94% 81% 100%
Benomyl 79% NT NT NT
Example 4
Treatment of Grape Phylloxera with Cinnamic Aldehyde and/or Coniferyl
Aldehyde alone and/or with Tween 80 and/or NaHCO.sub.3
Feeding Site Location Test
Mortality resulting from physiological process disruption is determined by
the Adult and Nymphal mortality rent and by the Egg Hatch experiment.
After hatching, new insects must secure a verified appropriate feeding
site. This activity must be successful if the life cycle of the insect is
to continue. Research indicates that approximately 80% of phylloxera
mortality occurs during this activity. Low dose concentrations of formulae
may be protective of grape stock roots by disrupting the "search and
identify feeding site" behavior of the insect. All three types of effects
are evaluated using the following protocols.
Adult and Nymphal Mortality Experiment
Approximately twenty four eggs of phylloxera were allowed to develop for up
to] 30 days on standard excised grape roots. At around 30 days, some of
the insects are nymphs while others are adults. New eggs were removed
during the process. Insect infected roots were submerged into a test
formula for 6 seconds then set aside to dry in the air. The percentage of
live insects, as defined by growth, oviposition or limb movement, was
determined after 5 days. An insect is considered dead if it abandons its
feed site. In an initial test, doses of 20,000 ppm cinnamic aldehyde in
water (i.e., 2% cinnamic aldehyde) with various additives were evaluated.
Cinnamic aldehyde without any additives produced 83.3% mortality. With 1%
Tween 80 added, 91.7% mortality was seen. With 6% NaHCO.sub.3 added to a
solution of 2% cinnamic aldehyde, 91.7% mortality was seen. With 1% Tween
80 and 6% NaHCO.sub.3 added to a solution of 2% cinnamic aldehyde, 100%
mortality was seen. Water with no additives produced no mortality, while a
positive control solution of 250 ppm malathion in water gave 100%
mortality.
Egg Hatch Experiment
Mixed age groups of 60 grape phylloxera eggs were established on filter
paper (Whatman #1, 5.5 cm circles) in 50.times.9 mm sealing plastic petri
dishes treated with 100 .mu.l of solution. A selected concentration of a
test formulation of 400 .mu.l was added to the filter disk and the dish
closed with the petri dish cover and placed in a plastic container box.
After 6 hours, the box was placed in an environmental chamber at
24.degree. C. in the dark. The eggs were placed in groups of 10. After one
week, the percentage of hatch is determined. In an initial test, doses
from 0.1 to 25,000 ppm cinnamic aldehyde in 6% NaHCO.sub.3, 2% Tween 80
were evaluated with a single group of eggs at each dosage. Three
replicates of the experiment were performed. The effects of the
formulation were evaluated after 7 days and scored as the number of nymphs
that died in the shell (DIS), or eggs that did not hatch completely (IH)
(i.e., all died). LD50 and LD95 were determined by probit analysis. At
5000 ppm cinnamic aldehyde, all nymphs died in the shell. At 100 ppm, 88%
died in the shell and the remaining 12% did not hatch completely. At 10
ppm, none died in the shell, but 100% of the eggs did not hatch
completely. The addition of 0.86 ml of a 10.degree. brix saponin solution
in water to the formulation at 100 ppm increased the number of nymphs
which died in the shell to 93%. Coniferyl aldehyde over the same dosage
range (in 6% NaHCO.sub.3, 2% Tween 80) was less potent. Although 10% of
nymphs died in the shell at 5000 ppm, at 1000 ppm, 12% died in the shell
and 85% hatched incompletely. All phylloxera eggs treated with H.sub.2 O
alone hatched; 100% of those treated with Carbofuran.RTM. (10 ppm) or
malathion (250 ppm) died in the shell.
Example 5
Protocol for Aphid and White Fly
Activity of cinnamic aldehyde and/or coniferyl aldehyde against black bean
aphid, Tetranychus urticae, and silverleaf white fly, Bemisia argentifolii
is determined as follows:
Petri Dish Bioassay: Aphids
Petri dishes (60 mm) were treated with a particular formulation (e.g.,
10-25,000 ppm) dissolved in water, and allowed to air dry. Twenty adult
aphids (Tetranychus urticae) were put in each dish, (replicate 10 times).
The mortality after three hours in contact with a treated plate was
compared to that of aphids in petri dishes treated only with diluent.
Malathion (250 ppm) was used as a positive control.
In initial experiments, cinnamic aldehyde at the indicated concentrations
in 2% Tween 80 and 6% NaHCO.sub.3 were tested. At concentrations of 2500
ppm and above, 100% of the aphids were killed. At 100 ppm and 10 ppm, 50%
and 25% mortality was observed. Twenty-five percent mortality was observed
with the concentration of 2% Tween 80 and 6% NaHCO.sub.3 (no aldehyde
added), and 100% of aphids were killed with malathion (250 ppm).
Plants are grown in 7.5 mm pot in potting soil in greenhouse. Cotton plants
are used for white fly and sugar beets are used for aphids. When plants
reach 3 leaf stage, they are infested with 60 of the specified anthropd (6
replications). The insect is allowed to settle and feed. The plant is
sprayed to runoff (about 5 ml) with a formulation containing 100 to 200
pm, or 0.1 to 2 g/l concentration of a test formulation. The plant is
covered with tall plastic cage (5 mm tall.times.10 mm diameter). The
mortality of the insects after three days on the plants sprayed with a
test formulation is determined and compared with that of insects on plants
sprayed only with water.
Petri Dish Bioassay: Silver Leaf White Fly
Petri dishes (60 mm) were treated with a test formulation (e.g., 10-25000
ppm) dissolved in water, and allowed to air dry. Twenty adult silver leaf
white fly were put in each dish, (replicate 10 times). The mortality after
three hours in contact with a treated plate, was compared to that of
silver leaf white fly in petri dishes treated only with diluent. Malathion
at 250 ppm was used as a positive control.
TABLE 5
Effect of Cinnamic Aldehyde and Coniferyl Aldehyde
Formulations on Aphid Mortality (Percent)
Aldehyde
Cinnamic
Cinnamic Coniferyl Aldehyde (10 g) +
Additive Aldehyde Aldehyde Coniferyl
Formulation None (20 g) (20 g) Aldehyde (10 g)
Percent Mortality
None 0 NT NT
T80 (10 g) NT NT
NaHCO.sub.3 NT NT
T80 + NaHCO.sub.3 25 98.6 NT NT
Saponin (1% 10 NT NT NT NT
brix)
Saponin + T80 NT NT NT NT
Saponin + NaHCO.sub.3 NT NT NT NT
Malathion 100 NT NT NT
H.sub.2 O (Neg. Control) .07 NT NT NT
In initial experiments, cinnamic aldehyde at the indicated concentrations
in 2% Tween 80 and 6% NaHCO.sub.3 were tested. At concentrations of 2500
ppm and above, 100% of the silver leaf white fly were killed. At 100 ppm
and 10 ppm, 50% and 25% mortality was observed. Twenty-five percent
mortality was observed with the concentration of 2% Tween 80 and 6%
NaHCO.sub.3 (no aldehyde added), and 100% of silver leaf white fly were
killed with malathion (250 ppm). See Table 6.
Example 6
Treatment of Nematodes
Various lands of nematodes infest plant tissue, including the stem and bulb
nematode (Ditylenchus dipsaci) and rootknot nematode (Meloidogyne spp.).
The treatment of stem nematode (Ditylenchus dipsaci) with various
formulations containing cinnamic aldehyde is tested as follows:
Stem nematodes
Stem nematodes are extracted from garlic cloves by chopping the tissue into
a mesh-bottomed beaker and suspending the mesh-bottomed beaker in a beaker
of water. Nematodes migrate from the host tissue and sink down through the
mesh into the bottom beaker. The supernatant water is removed and the
nematodes remaining in the beaker are transferred to a watchglass and used
in the treatment protocol as follows. Clear plastic trays are divided into
open-topped cells measuring 20 mm.times.20 mm.times.20 mm. One half ml of
tapwater at room temperature (19.degree. C.) is pipetted into each cell.
Ten nematodes are placed in each cell using an eyelash glued to a
dissecting needle to handle each animal. One-half ml of one test solution
is then added to each cell. Water is added to the control wells. Survival
of nematodes in the cell is monitored by observation using a binocular
microscope. The number of animals surviving 1, 5, 10, 20, 30 and 60
minutes after addition of the solutions is recorded. Mortality is assumed
if individual nematodes are immobile and fail to respond to manipulation.
The test is repeated three times.
TABLE 6
Effect of Cinnamic Aldehyde and Coniferyl Aldehyde
Formulations on Silver Leaf White Fly Mortality (Percent)
Aldehyde
Cinnamic
Cinnamic Coniferyl Aldehyde (10 g) +
Additive Aldehyde Aldehyde Coniferyl
Formulation None (20 g) (20 g) Aldehyde (10 g)
Percent Mortality
None 0 68.6 NT NT
T80 (10 g) 14.5 72.1 NT NT
NaHCO.sub.3 22.9 87.3 NT NT
T80 + NaHCO.sub.3 25.0 100 NT NT
Saponin (1% 10 NT NT NT NT
brix)
Saponin + T80 NT NT NT NT
Saponin + NaHCO.sub.3 NT NT NT NT
Malathion (250 100 NT NT NT
ppm)
H.sub.2 O (Neg. Control) 26.9 NT NT NT
In a double blind, concentrations of formula was tested for activity
against root-knot nematode, Meloidogyne javanica. Nematodes were put in
direct contact with the chemical and at 24 hour intervals, mortality was
assessed both visually and by probing. Meloidogyne javanica were produced
using hydroponics. The nematodes were harvested and used within 24 hours.
Approximately 100 nematodes in 0.07 mls of water were pipetted into a
syracuse dish (Fisher) and 1 ml of test formulation was immediately
pipetted into each dish. The dishes were then placed into plastic bags to
retain moisture and prevent evaporation. Four syracuse dishes were used
for each solution test formulation. Every 24 hours for 7 days, the
solutions were examined and the first 10 nematodes encountered were
assessed as either living or dead. This was based on morphological
integrity of the nematode and touch. Moving nematodes were counted as
living. As concentrations greater than 100 ppm cinnamic aldehyde in
vehicle (2% Tween 80, 6% NaHCO.sub.3), 100% nematodes were dead at 24
hours. At 10 ppm, 0%, 15%, 17.5% 22.5%, 27.5%, 52.5% and 52.5% were dead
at 24, 48, 72, 96, 108, 132, and 156 hours respectively. There was no
effect on mortality at 1 ppm and 0.1 ppm cinnamic aldehyde in vehicle.
Addition of a 1:60 dilution 10 brix concentrate of Yucca shidigera saponin
resulted in 100% mortality at 24 hours with the lowest concentration of
cinnamic aldehyde in vehicle tested, 0.1 ppm. However, saponin alone had
the same effect. EtOH (95%) killed all nematodes at 24 hours. Minimal
effect of the vehicle on mortality was observed: 2.5% at 72 hours and 5%
at 108 hours.
Example 7
Treatment of Strawberry Red Core (Phytophtora Fragariae)
Strawberry red core disease is caused by the fungus Phytophtora fragariae
Hickman which is spread by means of infected planting material or soil
infested with long-lived oospores of infected debris. Various formulations
containing cinnamic aldehyde and/or coniferyl aldehyde are tested as
follows: Macerated strawberry roots infected with Phytophthora fragariae
are thoroughly mixed with infested compost and allowed to decompose for 4
to 6 weeks to produce a well rotted inoculum for treatment. This is
divided into 1 kg lots and mixed with 1500 ml of a test formulation at
different concentrations. After 10 minutes treatment, the compost is
rinsed under running tap water on a 25 mm sieve for a minimum of 5 minutes
to remove all traces of the test formulation. The compost is then put into
9-cm diameter plastic pots and planted with 4 strawberry plants per pot.
Five pots are used for each treatment. Plants are grown in a controlled
environment room at 15.degree. C. and 18 h daylength; the compost is kept
damp to encourage infection. Pots are placed on grids to avoid cross
infection among treatments.
After 9 weeks the strawberry plant roots are washed free of compost and
examined for signs of infection by cutting roots longitudinally and
looking for red steles, and rotted or brown roots. All infections are
confirmed by microscope examination of root pieces for the presence of
oospores of Phytophtora fragariae.
Example 8
Stability of Cinnamic Aldehyde
Protocol to determine the stability of cinnamic aldehyde with and without
an anti-oxidant over time.
Cinnamic aldehyde at 2% by weight added to a formula containing 2% Tween 80
and 6% sodium bicarbonate with and without the addition of vitamin E
(tocopherol @ 1% of CNMA concentration).
The solutions to be maintained at 50.degree. C. for two weeks. The
solutions to be analyzed for cinnamic aldehyde concentration on regular
intervals during the two week period by HPLC/UV and recorded (high
pressure liquid chromatography ultra violet detection).
Example 9
Pitch Canker Disease
Pitch canker disease, caused by the fungus subglutinans f. sp. pini is
characterized by a resinous exudation on the surface of shoots, branches,
exposed roots and boles of infested trees. The host and geographic range
of the pitch canker pathogen has greatly increased since it was first
discovered in California in 1986. The pathogen has recently been
discovered in Mexico and Japan. An association of Engraver beetles
(Scolytidae: IPS species) as vectors of the Pitch Canker Fungus has been
made by Fox, et al., (1991).
Bioassay undertaken in double blind with Professor Tom Gordon, University
of California, Berkeley, using ProGuard PGXL formula and protocols.
Bioassay based on inhibition of radial growth of Fusarium subglutinans f
sp. pini.
Methods and Materials
1. 8 ml of product (concentration unknown) was pipetted into 200 ml of
molten 2% potato dextros agar (PDA), the mixture was dispersed into five
plastic petri dishes (25 ml dish).
2. Each of four plates was inoculated at the center with an agar plug
transferred from a growing PDA culture of Fusarium subglutinans f. sp.
pini (isolate SL-1, UCB). A fifth plate was left noninoculated as a
control.
3. Steps 1 and 2 were repeated for each formulation of the unknown.
4. Four PDA plates amended with 5 ppm benomyl were inoculated as described
in step 2.
5. Four unamended PDA plates were inoculated as described in step 2.
6. All inoculated and non-inoculated plates were incubated at 18.degree. C.
for five days, after which colony diameters were measured.
Results
Table 7 shows raw data averages for bioassay. Table 8 compares CNMA PGXL
formula, CNMA PGXL formula+saponin Yucca schidegira extract 10.degree.
BRIX) @ 0.86 ml, against controls.
TABLE 7
Radial Growth of Fusarium
Subglutinans f, sp. pini
(Data Averages)*
Treatment Colony diameter (cm)
PDA (unamended) 4.038
10 ppm CNMA 4.363
100 ppm CNMA 4.238
100 ppm CNMA + Saponin 4.300
Glutaraldehyde 2% 3.663
5,000 ppm CNMA + Saponin 2.913
10 ppm CNMA + Saponin 3.600
H.sub.2 O 3.738
2,500 ppm CNMA 3.513
2,500 ppm CNMA + Saponin 3.600
Saponin 4.138
5,000 CNMA 2.908
Formula Blank 4.380
12,500 CNMA + Saponin 0.000
25,000 CNMA 0.000
5 ppm Benomyl 0.000
(Positive Control)
12,500 ppm CNMA 0.000
25,000 CNMA + Saponin 0.000
TABLE 8
Radial Growth of F. Subglutinans f. sp. pini
Treatments
PGXL
PGXL CNMA + Saponin
CNMA (.86 ml)
ppm colony diameter (cm) colony diameter (cm)
10 4.36 4.09
100 4.24 4.3
2,500 3.51 3.6
5,000 2.98 2.91
12,500 0 0
25,000 0 0
Controls colony diameter (cm)
2% Glutaraldehyde 3.66
H.sub.2 O 3.74
5 ppm Benomyl 0
PDA (unamended) 4.04
Example 10
Overproduction of Aromatic
Aldehydes in Transgenic Plants
20 .mu.g of polyA RNA is prepared and cDNA synthesized. Part of this is
cloned into lambda-ZAP II vector (a commercially available cloning
vector). At least 500,000 recombinants are screened using an
oligonucleotide probe designed from conserved sequences of cloned CA4H and
CAD genes obtained from GenBank, or designed from peptide sequence of
purified protein from the intended host plant. Strongly hybridizing clones
are selected and used to rescreen the cDNA library. The resulting clones
are sequenced to enable the introduction of appropriate gene sequences
into a plant expression cassette in either antisense or sense orientation.
The antisense and sense constructs are introduced into Agrobacterium
tumefaciens LBA4404 by direct transformation following published
procedures. Tobacco (N. tabacum, variety Samsun) leaf discs are
transformed using well established published procedures (Horsch, et al.,
(1985 Science 227:1229-1231. Plants containing either CA4H or CAD
constructs are identified by PCR and selected for further analysis.
Plant material from both transformed and untransformed control plants is
used for determinations of CA4H and CAD enzyme activity using well
established published assays. Plants in which the activity of CA4H or CAD
has been reduced to less hand 20% of that seen in control plants are
selected for further analysis. Selected plants with low CA4H activity are
crossed with plants with low CAD activity and progeny inheriting both gene
construct are selected by PCR. Plants with suppressed CA4H and suppressed
CAD activity are analyzed for flavonoid aldehyde production using standard
published procedures.
Example 11
Production of Aromatic Aldehydes in Microbial Systems
A cDNA library is generated using RNA extracted from six week old tobacco
stems. 20 .mu.g of polyA RNA is prepared and cDNA synthesized. Part of
this is cloned into lambda-ZAP II vector (a commercially available cloning
vector). At least 500,000 recombinants are screened using an
oligonucleotide probe designed from peptide sequence sequences of CCoAr
protein purified from six week old tobacco stem tissue using the protocol
of Goffner, et al., Plant Physiol. (1994) 106:625. Strongly hybridizing
clones are selected and used to rescreen the cDNA library. The resulting
clones are sequenced to enable the identification of full-length cDNA
inserts and the introduction of appropriate CCoAR gene sequences into
yeast expression vector pMTL8110 (Faulkner, et al (1994) Gene 143:13-20.
The coding sequences for Rhodosporidium torulides phenylalanine ammonia
lyase (PAL; GenBank locus RHDPAL) and a parsley 4-coumarate:CoAl ligase
(4CL; GenBank locus PC4CL1AA) are similarly introduced into equivalent
yeast expression vectors. The PAL,4CL and CCoAR constructs are used to
transform Saccharomyces cerevisiae strains by electroporation using
established published procedures (Becker, and Guarente, Methods in
Enzymology 194:182-187, 1991; Simon, (1993) Methods in Enzymol
217:478-483. Transformants are selected on minimal medium lacking leucine.
Transformant strains carrying all three gene constructs are identified by
PCR and selecter for further analysis.
Extracts from both transformed and untransformed control strains are used
for determinations of PAL, 4CL and CCoAR enzyme activities using well
established published assays. Strains in which the activity of PAL, 4CL
and CCoAR is significantly greater than the background activity detected
in control strains are selected for further analysis. Selected strains are
analyzed for aromatic aldehyde production using standard published
procedures and those producing significant amounts of cinnamaldehyde are
selected for optimization of fermentation conditions.
As the above results show, potted roses or field grown roses sprayed to run
off with an emulsion containing cinnamic aldehyde and sodium bicarbonate
and concomitantly sprayed with saponin remained free of powdery mildew and
rust for up to 56 days as compared to plants sprayed only with water. The
plants also remained free of aphids. It has been reported that induced
systemic resistance to powdery mildew of roses sprayed with Rubigon
averages about 20 days. Mean disease control determinations of
approximately 70% were obtained for roses sprayed with an aqueous solution
of cinnamic aldehyde and coniferyl aldehyde or emulsions containing sodium
bicarbonate and cinnamic aldehyde and/or coniferyl aldehyde. In parallel
experiments, Benomyl gave a mean disease control of approximately 80%.
All publications and patent applications mentioned in this specification
are indicative of the level of skill of those skilled in the art to which
this invention pertains. All publications and patent applications are
herein incorporated by reference to the same extent as if each individual
publication or patent application was specifically and individually
indicated to be incorporated by reference.
The invention now having been fully described, it will be apparent to one
of ordinary skill in the art that many changes and modifications can be
made thereto without departing from the spirit or scope of the appended
claims.
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